Genetic Recombination and DNA Technology
is a process through
which the genetic makeup of an organism is altered or changed in some way.
This alteration may be favorable, undesirable, or have no direct effect
on an organism at all, depending upon the circumstances through which it
occurs. If recombination is favorable, it leads to genetic diversity
by insuring that the organism has the ability to express new characteristics
or traits which are not expressed by other members of the population, but
can be passed to other members or offspring. While recombination
is similar to mutation in that it results in a change in the genotype of
an organism, it is less likely to destroy the function of individual genes.
Homologous and Nonhomologous Recombination
Recombination can occur either in an homologous
or nonhomologous fashion. In eukaryotic cells, homologous recombination
occurs when the DNA which is exchanged is found in alternative forms of
the same gene, called alleles, which lie on homologous eukaryotic
chromosomes via the process of crossing over. Crossing over
occurs during the process of meiosis, when chromosomes line up. Occasionally,
chromatids from paired chromosomes overlap. Their DNA can then be
cleaved by endonuclease enzymes, then each piece swaps its place with the
other and is enzymatically ligated (joined) to the new chromatid.
When the pairs are separated from one another, each chromosome is now genetically
altered. In prokaryotic cells, homologous recombination occurs when
an endonuclease cleaves one DNA nucleoside leaving a free 3'-OH end, which
then acts as a primer for DNA synthesis to occur. The newly synthesized
DNA nucleoside bonds to a corresponding region of the bacterial chromosome
and forms a heteroduplex combination of single- and double-stranded
DNA. Such heteroduplex forms are catalyzed by enzymes which are coded
for by recombination (rec) genes.
Nonhomologous recombination does not
involve specialized rec enzymes, but allows for the the joining of DNA
molecules which have little similarity. Unlike homologous recombination,
this means that DNA fragments from one source, such as from a virus or
bacterial plasmid, can be incorporated into a bacterial chromosome or the
DNA of a eukaryotic cell. If the nonhomologous DNA comes from a virus,
a phenomenon called lysogenic conversion can occur, enabling the
host cell to express viral genes. For example, several bacteria species
such as Staphylococcus aureus, Clostridium botulinum,
and Corynebacterium diptheriae only become pathogenic after they
have been invaded by a virus.
Some cells contain small genetic elements
of about 1000 nucleotides which have the ability to move or "jump" from
one part of a chromosome to another. These elements, called insertion
sequences, do not appear to code for specific proteins but may serve
as regulators for recombination of genetic material at specific sites,
altering either promotor or structural genes. In her study of corn
genetics, Dr. Barbara McClintock discovered that other, larger transposable
elements, called transposons, serve as structural genes and code
for the production of specific proteins. The insertion sites for
these genes are random, thus their location can jump. Transposons are medically
important in that they may code for antibiotic resistance factors in bacteria,
and other microorganisms such as the pathogenic members of the protistan
genera Trypanosoma and Plasmodium utilize such genes to create
unique combinations of surface recognition glycoprotein compounds which
allow them to overcome host defenses.
Frederick Griffith demonstrated in 1928
that living unencapsulated, nonpathogenic Streptococcus pneumoniae
could become pathogenic if mixed with dead encapuslated pathogenic bacteria
of the same species. He called this process transformation,
but was unaware of the chemical composition of the genetic material which
was being passed between the dead and living cells. In 1944, Avery,
McCarty, and McCloud demonstrated that the material was DNA,
not protein or RNA, both of which were hypothesized to be primary information
storage molecules for cells. Later research would show that in order
for transformation to occur, the donor DNA must be released from a cell
or cells after lysis, and that the live recipient bacteria must be competent,
meaning that they must be capable of transporting the external DNA across
their own plasma membrane or have specific receptor sites on the cell surface
which enable them to do so. Also, before any recombination of the
donor and recipient DNA could occur, the recipient must be compatible with
the host, thus the highest frequency of recombination occurs in cells which
are closely related, such as different strains of the same species.
If tranformation does occur, the progeny of a transformed cell are said
to be recombinants. Examples of bacterial genera which can
undergo transformation include Acinetobacter, Bacillus, Haemophilus,
Neisseria, Rhizobium, Staphylococcus, and Streptococcus.
In 1952, Hershey and Chase demonstrated
that viruses could transfer DNA to host cells through the radioactive labelling
of bacteriophage protein and DNA. The process of viral-mediated DNA
exchange is called transduction. Since DNA viruses such as
some of the bacteriophages replicate within a host cell and host DNA is
fragmented enzymatically during this process, some of the host cell DNA
can be accidentally packaged by viral protein coats. After these
newly formed defective phages are released from the original host
bacterium, they can infect new host cells and deliver their DNA, where
recombination can take place. This is not fatal to the host, and
can confer upon it new characteristics. If the genes carried by the
defective phage are homologous to those of the recipient, the process is
termed generallized transduction, and the recipient is said to be
transduced. If the genes are not homologous, such as those genes
carried by temperate phages which are not defective, they may become incorporated
into the bacterial chromosome. If this DNA is copied from the host
chromosome during the subsequent lytic cycle, it may also contain some
of the host DNA, which can then be delivered to a new host. This
process is called speciallized transduction, and can result in the
lysogenic conversion of species such as Corynebacterium diptheriae
and Staphylococcus aureus.
Plasmids and the Process of Conjugation
In 1950, Lederburg and Tatum discovered
that some bacteria contain small, extrachromosomal genetic elements called
plasmids which store genetic information not found in the chromosome.
Plasmids generally do not contain information which is necessary for the
essential metabolic activities of the cell; however, they do contain genes
which can determine the ability of the organism to pass genetic material
to recipient cells. Lederburg and Tatum found that some strains of
E. coli contain the F (fertility) plasmid which enables
these cells to form sex or conjugation pili. Later, it was found
that plasmids carry other characteristics as well. E. coli,
Shigella sp. and Salmonella sp. all have plasmids called
R (resistance) factors which can give the cell the ability
to produce enzymes which confer resistance to some antibiotics. Some
strains of E. coli also carry colicinogenic plasmids which
code for the synthesis of colicin, a toxin which kills competing strains
of E. coli. Plasmids also can give bacteria the ability to
produce toxins, and to degrade complex organic compounds such as hydrocarbons.
If a bacterium has an F-plasmid, it is referred
to as an F+ donor strain. If the plasmid is not present, the
bacterium is called an F- recipient. During conjugation, The
F+ cell forms a conjugation pilus between itself and the recipient,
which links the two cells and allows the commingling of their cytoplasm.
The F- plasmid is replicated within the donor, then passes through the
pilus to the recipient, which now becomes an F+ cell. If the donor
plasmid recombines with the donor chromosome, a new type of cell called
an Hfr (High frequency of recombination) cell
is formed. This cell can also participate in conjugation, however,
a portion of the recombined chromosome is passed through the conjugation
tube to the F- recipient. After recombination, the F- cell is converted
into a new Hfr cell. The new cell, however, is generally will be
incapable of conjugation itself, since only a portion of the genes contained
in the original plasmid are passed via this process.
Another phenomenon which can occur involves the formation of a
new plasmid composed of genes which were located in the Hfr chromosome.
This plasmid contains both chromosomal genes and recombined plasmid genes.
After the new plasmid forms, the cell is called an F' cell rather than
an Hfr cell.
Recombinant DNA Technology
Recombinant DNA technology involves the
deliberate artificial union of DNA molecules from two or more different
sources in a nonhomologous fashion. This process allows researchers
to give unrelated organisms the ability to express new phenotypes for a
variety of different purposes. For example, some individuals suffer
from diabetes, which is the inability to control the level of sugar in
the bloodstream. This disorder arises from the lack of the hormone
insulin, which is produced by special cells in the pancreas. In past
years, insulin used in the treatment of diabetes was derived from animals,
since it was impossible to synthesize sufficient quantities of human insulin
(humulin). Unfortunately, many individuals suffered hypersensitive
reactions to animal insulin. With the advent of new molecular techniques
for the insertion of foreign DNA into a bacterial chromosome or plasmid,
it became possible to produce large quantities of humulin. In brief,
human insulin is composed of two polypeptides coded for by two different
genes. Researchers inserted these genes into the lac operon of E.
coli, just after a nucleotide triplet which codes for the amino acid methionine.
This recombined bacterium began to produce humulin when in the presence
of lactose, since the lac operon controls the production of enzymes necessary
for the transport and catabolism of this sugar.
The ability to insert foreign gene sequences
into nonhomologous host DNA is derived from scientists' knowledge of a
group of enzymes produced by some bacterial species as a protective measure
against viral infection. These enzymes, called restriction endonucleases
cleave (cut) DNA along specific sequences of nucleotides.
If DNA is cleaved in such a way as to leave a few bases overhanging in
palindromic sequences (sequences which read the same way in both
directions; such as GGCATACGG and CCGTATGCC), the piece of DNA is said
to have "sticky ends" since these overhanging portions of the 5' and 3'
ends will form bonds (anneal) with any segment of DNA which has been cleaved
to produce overhanging segments with complementary base pairs. If,
however, the restriction enzyme cleaves the molecule in such as way as
to leave corresponding 5' and 3' ends bonded together in complementary
fashion, the cut DNA molecule is said to have "blunt ends". To bind
a new or donor DNA fragment, it is necessary to establish artificial palindromy
by binding multiple adenine (poly A) nucleotides to the donor and
multiple thymine (poly T) to the recipient.
Another technique which can be used to produce
suitable DNA sequences for genetic engineering purposes is to utilize the
properties of a viral enzyme called reverse transcriptase.
One of the problems inherent in the engineering of eukaryote DNA is that
of split genes. Recall that when transcription occurs in the eukaryotic
nucleus, the usable portions of DNA, called exons, must first be separated
from the nonsense portions called introns. Rather than attempting
to perform this process in-vitro (outside of the body), scientists purify
the completed mRNA, then treat it with reverse transcriptase. This
enzyme binds DNA nucleotides to the mRNA template, producing a new DNA
nucleoside. DNA polymerase is used to complete the complementary
nucleoside. The newly formed gene can then be inserted into a bacterial
The Polymerase Chain Reaction
Sometimes it is necessary to have multiple copies of
a sequence of DNA, if, for example, a sample of blood, semen, mtDNA (mitochondrial
DNA), or some other DNA-containing substance is too small for proper analysis
or genetic manipulation. The technique called the polymerase chain
reaction (PCR) can be used to amplify (clone) large
numbers of the same DNA sequence. To perform PCR, a piece of DNA
is extracted from a sample and purified. This DNA is then heated
to a temperature between 90o and 100o C, which causes
the hydrogen bonds between complementary nucleosides to break without breaking
the phosphodiester bonds between nucleotides. Short segments of artificially
produced DNA called oligonucleotides, which are complementary to
the DNA nucleosides to be amplified, are added to the heated DNA sample,
which is then allowed to cool. These bind, or anneal, to the original
DNA nucleosides. Oligonucleotides act as primers for DNA replication,
which is stimulated by the addition of individual nucleotides and DNA polymerase.
This process is repeated several times (called cycles), with each
cycle resulting in a doubling of the number of identical copies of the
original piece of DNA. If PCR is performed properly, over one million
copies of the orignal DNA strand can be produced within an hour.
Cloning is the asexual reproduction of
genes containing recombined DNA. Plasmids containing recombined gene
sequences are often used as vectors for this technique, since they can
be easily engineered and owing to the high reproductive rates of many bacterial
species. To clone a sequence of DNA, a vector is prepared containing
the sequence along with another gene, which codes for the production of
an enzyme for resistance to a particular antibiotic. The vector is
then inserted into bacterial cells via transformation or transduction,
and the newly transformed or tran sduced cells are placed on media containing
the antibiotic. As the bacteria reproduce, many copies of the DNA
sequence can be guaranteed, since only those cells resistant to the antibiotic
will survive on the medium. However, if foreign genes have been inserted
by some other means such as unwanted viral infection or conjugation with
other bacteria at the site of antibiotic resistance, the ability to block
the antibiotic is lost. Such insertional inactivation is a useful
means to determine if foreign DNA is present in transformed cells.
If the researcher wishes to recombine the
entire DNA code (genome) from two or more cells, the technique of protoplast
fusion can be performed. To fuse prokaryote cells, the bacteria
must first be stripped of their cell walls, then stored in a buffer solution
which contains a high concentration of a solute such as sucrose.
The buffer prevents the protoplast cells from lysing due to osmotic pressure
differences inside and outside of the cell membrane. Protoplasts
are then treated with polyethylene glycol which causes them to fuse.
The chromosomes of the fused cells then recombine.
This technique can also be used with eukaryotic
cells. For example, cancer cells called mylenomas can be extracted
from mouse tumor tissue. These mutant tumor cells replicate rapidly
(hyperplasia) and grow much larger than healthy cells of the same tissue
type (hypertrophy). Mylenoma cells can be fused in-vitro with antibody-
producing B plasma cells, producing a new cell type called a hybridoma.
Hybridoma cells can be artificially cultured, grow quickly, and produce
large quantities of specific immunoglobulins, called monoclonal antibodies,
which when purified can be used to stimulate artificial active humoral
immunity in patients suffering from various viral and bacterial- induced
diseases. This technique shows great promise in the treatment of
many current human diseases.
Critical Thinking Question
You and your research associates have just isolated
a rare gene in a species of sea urchin (a multicellular eukaryotic marine
animal) which codes for the production of an enzyme that blocks the entry
of HIV into human cells. Unfortunately, you have two problems; (A)
you have isolated only a small sample of the genetic material from the
one urchin you have been able to collect, and (B) gel electrophoresis has
shown the sequence of DNA you need to be full of intron material.
Describe how you could overcome these problems, as well as how you could
produce sufficient quantities of the enzyme to begin drug trials with HIV
positive and full-blown AIDS patients.
Test Yourself- Use this to test yourself
about comcepts associated with recombination.